There has been a renewed interest in the development of better hemostatic agents for traumatic injuries. (1, 2). As a result of their hemostatic and adhesive properties, sealants, and particularly fibrin sealants are extensively used today in most surgical specialties to reduce blood loss and post-operative bleeding because of the ability to adhere to human tissue as it polymerizes (3,4). These compounds are used to seal or reinforce wounds that have been sutured or stapled; they can also be used with or without pressure over an injured area.
All available fibrin sealants are being produced by mixing fibrinogen with thrombin in different mediums, concentrations and additives such as Factor XIII. These techniques are prone to produce autoimmune reactions, in addition to severely limiting the activity and shelf life of components; fibrinogen is degraded by proteolysis and thrombin tends to cleave itself when is in solution. For this reason, fibrin components are often provided in lyophilized form, with freezing requirements and short shelf life. They must be used with sponges and/or films that are not convenient in trauma procedures.
In the process of developing a fibrin sealant for use in cases of non-compressible hemorrhage (ClotFoam application patent Ser. No. 12/419,734) it was determined that a fibrin monomer in acetic acid ready to polymerize at change of pH, offers substantial advantages for use in cases of non-compressible hemorrhage over cleavage of fibrinogen by thrombin, as generally used today. The advantages of incorporating a fibrin monomer in the sealant formulation are further described below.
Fibrin Sealants Current and Limitations.
Several products are commercially available (e.g., Tissel, Evicel, Gelfoam, Floseal, etc) [5-7], however, these products have significant limitations which have prevented their widespread use in trauma, emergency medicine and laparascopic surgery; because current haemostatic agents require compression. In addition, all sealants expose thrombin to the immune system with the consequently risk of autoimmune disease, as well as other anaphylactic reactions due to plasma proteins. Furthermore the incorporation of fibrinogen and thrombin in solution exposes the sealant to a rapid proteolitic degradation and self cleavage.
Our Alternative Approach: Fibrin monomer produced by the methods described bellow is designed to be used as sealant component for that polymerizes from a fibrin monomer at a change of pH. The Monomer produced by this method is embedded in a scaffold and neutralized by components of this scaffold to achieve hemostasis.
The scaffold (e.g. CloFoam) presents to tissues fibrin monomer in acetic acid at a concentration of 12 mg/ml solution, which is embedded in an hydrogel that polymerizes by a change of pH, and that is rapidly stabilized by Factor XIII.(8) 
The fibrin monomer delivered as a ready-to-polymerize fibrin in solution bypasses the fibrinogen cleavage process. When brought to neutral pH the polymerization of monomer and the following stabilization of the polymer is so rapid that the fibrin matrix forms in a matter of seconds, bonds with tissues in the midst of flowing blood, and remains at the lacerated site to form a clot. By including a fibrin monomer in acid solution rather than thrombin and fibrinogen, the sealant has a longer shelf life, better adhesion and avoids exposing thrombin to the immune system.
Advantages of Polymerization of Fibrin Monomer Over Fibrinogen Polymerization by Thrombin. Interactions Between Fibrin and Proteins
Several hemostasis proteins, such as tissue-type plasminogen activator, plasminogen and FXIII, bind to fibrin. The fibrinous matrix of a wound also contains other plasma proteins, such as fibronectin and vitronectin. Fibronectin and vitronectin may act as a bridge molecule between smooth muscle cells and fibrin by binding to the a5 b1 or avb3 integrin receptor of cells. [9] In addition, fibronectin also binds fibrin exclusively through the aC-domain of the latter. This binding site is not accessible in fibrinogen, but becomes exposed in fibrin. Vitronectin directly associates with fibrin.
Thus, fibrin functions as bridging molecule for many types of cell-cell interactions and provides a critical provisional matrix at sites of injury. Fibrin-coated matrices have been reported to bind EC, smooth muscle cells, keratinocytes, fibroblasts, and leukocytes. These cells can bind directly to fibrin via cell surface integrin receptors and non-integrin (e.g. VE-Cadherin, ICAM-1, P-selectin, and GPIba) receptors. [10]. Integrins, transmembrane cell adhesion molecules that consist of an alpha and beta subunits, have been demonstrated to bind to fibrin, and are aMb2 on leukocytes, allbb3 on platelets and avb3, avb5 and a5 b1 on EC and fibroblasts.
Clot retraction by nucleated cells is very important for proper wound healing [11]. Binding of a5b1 integrin to fibrin in the clot promotes the retraction of the clot and changes the shape of the cell [12]. The contribution of the avb3 integrin to clot retraction during vascular healing has been demonstrated in many studies as well as the involvement of the allbb3 and aMb2 integrins [13]
Integrins facilitate the binding of EC to ECM proteins. The Aalpha chain of fibrinogen contains RGD sequences at positions 95-97 and 572-574, The Aalpha 572-574 RGD sequence binds the avb3 integrin in humans. The Aalpha572-574 RGD sequence is also required for the interaction of fibrinogen with a5b1 integrin [14], which plays an important role in cell adhesion. However, these observations may be influenced by the albumin molecule that is bound to the free sulfhydryl-group of the truncated Aalpha chain of fibrinogen Nieuwegein molecule, which may affect the fibrin structure, and endothelial invasion, and tube formation in the fibrin matrix.
Fibrin can also stabilize the expression of avb3-integrin on cultured human microvascular EC and therefore promote migration of these cells on provisional matrix proteins. ECs interact with fibrin via a number of receptors, such as Inter-Cellular Adhesion Molecule 1 (ICAM-1), VE-Cadherin, CD-44, and integrins. It has been observed that ICAM-1 binds the 117-133 sequence on the fibrinogen gamma chain. The beta15-42 sequence on fibrin plays an important role during the process of neovascularization [15]. It has been demonstrated that a fragment corresponding to the first four extracellular domains of VE-cadherin (cadherin 5) binds to this sequence. The fibrin(ogen) Aalpha572-574 RGD sequence that binds integrin avb3 and a5b1, plays a significant role during angiogenesis.
The power to stick to the lacerated tissue in a pool of blood also depends on the cellular and matrix interactions. The characteristics of the fibrin itself, such as the thickness of fibers, number of branch points, porosity, permeability and other polymerization characteristics define the interactions between specific binding sites on fibrin, pro-enzymes, clotting factors, enzyme inhibitors, and cell receptors [24]. The structure of the fibrin matrix affects its biological function. For example, more coarse matrices show a faster fibrinolysis and the pH of the fibrin matrix determines the in-growth of tubular structures. Opaque matrices at pH 7.0 consist of thick fibers and tube formation proceeds at a faster rate than in transparent matrices at pH 7.8 that consists of thinner fibers [20]. Several conditions may affect fibrin structure, such as the clotting rate (can be modulated by concentration of thrombin and salt content), but also by the presence of metal ions, proteins and enzymes, the rate of polymerization (determined by FXIII concentration and FXIII activation rate), and the rate of lateral polymerization (affected by fibrinopeptide B release and cross-linking sites on alpha and gamma chains). Chloride ions have been identified as modulators of fibrin polymerization, because these ions control fiber size by inhibiting the growth of thicker, stiffer, and straighter fibers.
Cost-effective methods of producing fibrin monomer: The preparation, properties, polymerization, equilibria in the fibrinogen-fibrin conversion, solubility, activation and crosslinking of fibrin monomer has been studied by several authors since 1968 (16-23). An experimental method for producing fibrin monomer was first described and published by Belitser et al (1968, BBA) (24) This method limits the production of monomer to a few milligrams per day.
Although U.S. Pat. No. 5,750,657 to Edwardson et al. describes a method of preparing a fibrin sealant utilizing a fibrin monomer composition, the ClotFoam sealant composition and use to which the production method of fibrin monomer herewith described is entirely novel and allows to produce the monomer in a cost-effective manner.